利用RAPD对稻蝗属昆虫亲缘关系的研究

通过20个随机引物的PCR扩增,得到了日本主要稻蝗的随机扩增多态性DNA（RAPD）图谱,根据扩增结果,计算了种间相似系数和遗传距离,建立了UPGMA系统树。结果表明,分布没有重叠、种间容易交配、能产生杂种的中华稻蝗台湾亚种与小翅稻蝗的亲缘关系最近;分布重叠的日本稻蝗与中华稻蝗台湾亚种、日本稻蝗和小翅稻蝗的亲缘关系较近。小稻蝗与其它3种稻蝗的亲缘关系较远。     

山稻蝗及相关物种Cyt b基因序列及其遗传关系

中国7个不同地理区域的山稻蝗Oxya agavisa共16个个体的线粒体DNA细胞色素b基因中间部分序列被测定,分析和比较,同时与小稻蝗O.inricata,日本稻蝗O.japonica和中华稻蝗O.chinensis相应序列进行比较,用瘤锥蝗科的曲尾似橄蝗Pseudomorphacris hollisi和蚱科的日本蚱Tetrix japonica作外群,构建了山稻蝗不同单倍型及其相关物种的NJ分子系统树。在获得的山稻蝗432bp的序列中,A＋T约占71．0％,其中9个核苷酸位点存在变异（约占所测核苷酸的2．08％）,但权有1个位点引起了氨基酸的变异,就每个氨基酸密码子来看,第三位点的A＋T含量较,达88．3％。分子系统树显示,山稻蝗6个单倍型聚为一个簇,彼此之间有一定的分歧,其分枝与地理分布没有直接的关系;该种与日本稻蝗关系较近,与中华稻蝗和小稻蝗相对较远。并对此进行了讨论。     

A molecular phylogeny of Oxya (Orthoptera: Acridoidea) in China inferred from partial cytochrome b gene sequences

The grasshoppers of the genus Oxya are well known to damage rice, sugar cane, and other crops, yet their phylogenetic relationships have not been examined with molecular data. In this study, we obtained the 432 bp DNA sequences of the mitochondrial cytochrome b gene from 91 individuals of nine Oxya species and two outgroups (Gesonula punctifrons and Acrida cinerea). Phylogenetic analyses for the molecular data set were then carried out using the maximum parsimony and neighbor-joining methods. The results showed that the nine Oxya species form four well-supported clades, which include (1) O. intricata and O. flavefemura; (2) O. japonica and O. bicingula; (3) O. agavisa; and (4) O. chinensis, O. brachyptera, O. adentata, and O. hainanensis, respectively. In particular, the monophyly of O. hainanensis and O. agavisa is strongly supported, respectively. However, O. flavefemura and O. intricata, O. bicingula, and O. japonica form paraphyletic groups, respectively, and O. chinensis, O. adentata, and O. brachyptera form a polyphyletic group, suggesting that they should be merged as few as three species.     

Molecular cloning and characterization of novel centromeric repetitive DNA sequences in the blue-breasted quail (Coturnix chinensis,Galliformes)

A new family of centromeric highly repetitive DNA sequences was isolated from EcoRI-digested genomic DNA of the blue-breasted quail (Coturnix chinensis, Galliformes), and characterized by filter hybridization and chromosome in situ hybridization. The repeated elements were divided into two types by nucleotide length and chromosomal distribution; the 578-bp element predominantly localized to microchromosomes and the 1,524-bp element localized to chromosomes 1 and 2. The 578-bp element represented tandem arrays and did not hybridize to genomic DNAs of other Galliformes species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and guinea fowl (Numida meleagris). On the other hand, the 1,524-bp element was not organized in tandem arrays, and did hybridize to the genomic DNAs of three other Galliformes species, suggesting that the 1,524-bp element is highly conserved in the Galliformes. The 578-bp element was composed of basic 20-bp internal repeats, and the consensus nucleotide sequence of the internal repeats had homologies to the 41-42 bp CNM repeat and the XHOI family repeat of chicken. Our data suggest that the microchromosome-specific highly repetitive sequences of the blue-breasted quail and chicken were derived from a common ancestral sequence, and that they are one of the major and essential components of chromosomal heterochromatin in Galliformes species.     

Characterization and chromosomal distribution of a novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica)

A novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica) was isolated from genomic DNA digested with restriction endonuclease, Bg/II. Sequence analysis of three different-size clones revealed the presence of a tandem array of a GC-rich 41 bp repeated element. This sequence was localized by fluorescence in situ hybridization (FISH) primarily to microchromosomes of Japanese quail (2n = 78); approximately 50 of the 66 microchromosomes showed positive signals, although hybridization signals were also detected on chromosomes 4 and W. This satellite DNA did not cross-hybridize with genomic DNA of chicken (Gallus gallus) and Chinese painted quail (Excalfactoria chinensis) under moderately stringent conditions, suggesting that this class of repetitive DNA sequences was species specific and fairly divergent in Galliformes species.     

九种蝗虫核型似近系数的聚类分析研究

应用核型似近系数聚类分析方法,研究了小稻蝗Oxya hyla intricata、山稻蝗O.Agav-isa、上海稻蝗O.Shanghaiensis无齿稻蝗O.Adentata、中华稻蝗O.Chisensis、日本稻蝗O.Japonica、短额负蝗Atractomorpha sinensis、奇异负蝗A.Pergrina和日本蚱Tetrix japonica等9种蝗虫的亲缘关系。结果显示,9种蝗虫分为3类：稻蝗,负蝗和蚱。6种稻蝗之间的核型似近系数(λ)在0.961～0.5695之间,2种负蝗的λ＝0.5867,日本蚱与这8种蝗虫的λ在0.5318～.0322。聚类图直观地反映出它们的亲缘关系与形态分类学的分类结果相一致。从9种蝗虫 的核型演化上看,日本蚱是较原始的类型,负蝗分化也较早,而稻蝗则是较进化的类型。     

Centromeric tandem repeat from the chaffinch genome: isolation and molecular characterization.

A new family of avian centromeric satellites is described. The highly repeated sequence, designated FCP (Fringilla coelebs PstI element), was cloned from the 500-bp PstI digest fraction of the chaffinch (Fringilla coelebs L.) genomic DNA, sequenced, and characterized. The FCP repeat was found to have 505-506 bp length of monomer, 57% content of GC, to compose about 0.9% of the chaffinch genome, and to be highly methylated. Results of Southern-blot hybridization of cloned FCP element onto genomic DNA digested with different restriction enzymes, and sequencing directly from total genomic DNA using FCP-specific primers and ThermoFidelase enzyme (Fidelity Systems Inc.) were in agreement with a tandem arrangement of this repeat in the chaffinch genome. Five positions of single-nucleotide polymorphism (SNP) were found in the FCP monomers using direct genomic sequencing. Fluorescence in situ hybridization (FISH) with FCP probe and primed in situ labelling (PRINS) with FCP specific primers showed that the FCP elements occupy pericentric regions of all chaffinch chromosomes. On chromosome spreads, the fluorescent signals were also observed in the intercentromeric connectives between nonhomologous chromosomes. The results suggest that the centromeric FCP repeat is responsible for chromosome ordering during mitosis in chaffinch.     

Genomic Subtraction Recovers Rye-Specific DNA Elements Enriched in the Rye Genome

Repetitive DNA sequence families have been identified in methylated relic DNAs of rye. This study sought to isolate rye genome-specific repetitive elements regardless of the level of methylation, using a genomic subtraction method. The total genomic DNAs of rye-chromosome-addition-wheat lines were cleaved to short fragments with a methylation-insensitive 4-bp cutter, MboI, and then common DNA sequences between rye and wheat were subtracted by annealing with excess wheat genomic DNA. Four classes of rye-specific repetitive elements were successfully isolated from both the methylated and non-methylated regions of the genome. Annealing of the DNA mixture at a ratio of the enzyme-restricted fragments:the sonicated fragments (1:3–1:5) was key to this success. Two classes of repetitive elements identified here belong to representative repetitive families: the tandem 350-family and the dispersed R173 family. Southern blot hybridization patterns of the two repetitive elements showed distinct fragments in methylation-insensitive EcoO109I digests, but continuous smear signals in the methylation-sensitive PstI and SalI digests, indicating that both of the known families are contained in the methylated regions. The subtelomeric tandem 350-family is organized by multimers of a 380-bp-core unit defined by the restriction enzyme EcoO109I. The other two repetitive element classes had new DNA sequences (444, 89 bp) and different core-unit sizes, as defined by methylation-sensitive enzymes. The EcoO109I recognition sites consisting of PyCCNGGPu-multi sequences existed with high frequency in the four types of rye repetitive families and might be a useful tool for studying the genomic organization and differentiation of this species.     

五种稻蝗染色体核型和C带带型的比较

本文研究了五种稻蝗Oxya chinensis、O. shanghaiensis、O. adentata、O. hyla intricara、O. agavisa的染色体核型和C带带型.玻片制备采用压片法,BSG法C带处理.结果表明:五种稻蝗染色体核型差异较小,具有属的保守性,但在C带方面则显示出一定的差别,种间染色体带型的差异大小与形态学上的差异程度具明显的正相关.其中小稻蝗和山稻蝗各自具有独特的带型结构,与其余三种显然不同,而中华稻蝗、上海稻蝗、无齿稻蝗虽在主要标志性染色体上带纹趋于一致,但在一些细微结构和统计数据方面存在一定差异.本文对五种稻蝗减数分裂中期Ⅰ的染色体数目和交叉定位也作了统计和比较,在染色体行为分析方面做了初步工作.据此,本文讨论了五种稻蝗的染色体分类问题.     

云南三种稻蝗基因组DNA的RAPD多态性分析

应用RAPD技术对采自云南龙陵两交水和腾冲曲石马鹿冲的24头稻蝗进行了基因组DNA多态性分析,10条随机引物共产生129条清晰、稳定的谱带.基于Jaccard相似系数,用Between-groups Linkage法得出的聚类图与基于Nei's遗传距离分别用UPGMA和NJ法构建的分子系统树基本一致:供试的所有稻蝗个体分为3个聚类簇:龙陵两交水的3、7～8号个体与腾冲曲石马鹿冲(海拔1450m)的8个个体聚为一支,龙陵两交水的1、2、4～6号个体聚为另一支,两支相聚后与腾冲曲石马鹿冲(海拔1 550 m)的8个个体聚为一起.形态学鉴定表明:龙陵两交水的3、7～8号个体为日本稻蝗,1、2、4～6号个体为小稻蝗,腾冲曲石马鹿冲(海拔1450 m)的8个个体均为日本稻蝗,腾冲曲石马鹿冲(海拔1550 m)的个体为有待进一步鉴定的未知种.形态学鉴定结果与聚类结果完全吻合,充分展示了RAPD技术在区分近缘物种方面的独到优势.比较不同采集地的日本稻蝗、小稻蝗的RAPD分析结果,发现云南的这两种稻蝗在同物种不同种群中的遗传多样性最为丰富,从一个侧面显示出我国云南昆虫的物种多样性及物种内部丰富的遗传多样性.     

Satellite DNA in the elm leaf beetle,Xanthogaleruca luteola (Coleoptera,Chrysomelidae): characterization,interpopulation analysis,and chromosome location

In this paper the satellite DNA (stDNA) of the phytophagous beetle Xanthogaleruca luteola is analyzed. It is organized in a tandem repeat of 149-bp-long monomers, has an AT content of 59%, and presents inverted internal repeats. Restriction analysis of the total DNA with methylation-sensitive enzymes suggests that this repetitive DNA is not methylated. Analysis of the electrophoretic mobility of stDNA on non-denaturing polyacrylamide gels showed that this stDNA is not curved. In situ hybridization with a biotinylated probe of the stDNA revealed a pericentromeric localization of these sequences in the majority of the meiotic bivalents. We have studied the stDNA of X. luteola from two populations with very distinct geographical origins. The sequence and phylogenetic analysis of monomers from these two populations showed that the repetitive element is conserved within the species. Putative gene conversion tracts are identified when the different monomers of the same population are compared. These results could indicate the existence of processes of homogenization that would extend these mutations to all the satellite repeats.     

CYTOTAXONOMIC STUDY OF OXYA SPECIES IN CHINA (ORTHOPTERA: ACRIDOIDEA)

Abstract  The chromosomal conventional karyotype and C-banding karyotype of eight species in the genus Oxya (Catantopidae) are analysed. The result shows that O. chinensis (Thunberg), O. shanghaiensis Willemse and O. adentata Willemse all have similar C-banding distribution, but the model of chromosomal group, the chiasma localization data and the total heterochromatin content (THC) value are different. O. agavisa Tsai has its own c-banding feature and is distinguished from other species in the genus. O. bicingula Ma et al . is similar to both the O. chinensis group and O . a gavisu in chromosomal marks and morphological characters, indicating that these three species have some relations during the evolutionary process. O. hyla intricata (Stal) is a "sibling species group" as indicated by the variation of the morphological feature and there are also diversiforms in cytotaxonomic marks. It seems that this group has higher differentiational speed and the speciational evolution is more active in modern times. In this group, O. apicocingula sp. nov. and O. flave femura sp. nov. are more specialized than the other populations which have not many terminal C-bands in genome. As a conclusion, we consider that the evolutionary rate of the species in Oxya genus is unbalanced. This status is due to the actions from both the hereditary basis and the environmental condition of the different species.     

A direct repeat sequence associated with the centromeric retrotransposons in wheat.

A high-density BAC filter of Triticum monococcum was screened for the presence of a centromeric retrotransposon using the integrase region as a probe. Southern hybridization to the BAC digests using total genomic DNA probes of Triticum monococcum, Triticum aestivum, and Hordeum vulgare detected differentially hybridizing restriction fragments between wheat and barley. The fragments that hybridized to genomic DNA of wheat but not to that of barley were subcloned. Fluorescence in situ hybridization (FISH) analysis indicated that the clone pHind258 hybridized strongly to centromeric regions in wheat and rye and weakly to those in barley. The sequence of pHind258 was homologous to integrase and long terminal repeats of centromeric Ty3-gypsy retrotransposons of cereal species. Additionally, pHind258 has a pair of 192-bp direct repeats. FISH analysis indicated that the 192-bp repeat probe hybridized to centromeres of wheat and rye but not to those of barley. We found differential FISH signal intensities among wheat chromosomes using the 192-bp probe. In general, the A-genome chromosomes possess strong FISH signals, the B-genome chromosomes possess moderate signals, and the D-genome chromosomes possess weak signals. This was consistent with the estimated copy numbers of the 192-bp repeats in the ancestral species of hexaploid wheat.     

Onchocerca volvulus: application of the polymerase chain reaction to identification and strain differentiation of the parasite

Previous studies have demonstrated that the genome of Onchocerca volvulus contains a variable tandemly repeated DNA sequence family with a unit length of 150 bp. The variability of the 150-bp family has been exploited to develop O. volvulus strain and species specific DNA probes. Application of these DNA probes to the study of the epidemiologically most significant life cycle stages of the parasite has been confounded by several obstacles. These include the relative insensitivity of some of the DNA probes and the difficulty in releasing genomic DNA from infective larvae and skin microfilariae in a form that may be directly detected by hybridization to the probes. DNA sequence comparison of 18 known examples of the 150-bp repeat has been used to develop two populations of degenerate oligonucleotides. These oligonucleotides have been shown to support the amplification of the 150-bp repeat family from Onchocerca DNA, using the polymerase chain reaction. Both strain and species specific members of the repeat family are faithfully amplified, allowing characterization of a parasite on the basis of hybridization of the PCR amplification products to the previously developed DNA probes. This method is shown to be applicable to all diagnostically important forms of the parasite, including adults, infective larvae, and skin microfilariae. In addition, the method is capable of detecting O. volvulus infective larvae directly in extracts of blackfly vectors.     

Chromosomal localization of a novel repetitive sequence in theChenopodium quinoa genome

In this study, a novel repetitive sequence pTaq10 was isolated from theTaq I digest of the genomic DNA of the pseudocerealChenopodium quinoa. Sequence analysis indicated that this 286-bp monomer is not homologous to any known retroelement sequence. FISH and Southern blot analysis showed that this sequence is characterized by an interspersed genomic organization. After FISH, hybridization signals were observed as small dots spread throughout all of the chromosomes. pTaq hybridization signals were excluded from 45S rRNA gene loci, but they partly overlapped with 5S rDNA loci. pTaq10 is not a species-specific sequence, as it was also detected inC. berlandieri.     

Distribution and sequence analysis of the centromere-associated repetitive element CEN38 of Sorghum bicolor (Poaceae)

Fluorescence in situ hybridization (FISH) of a large-insert genomic clone, BAC 22B2, previously suggested that Sorghum bicolor (2n = 20) has the tetraploid architecture A(b)A(b)B(b)B(b). Here, we report on BAC 22B2 subclone pCEN38 (1047-bp insert) as related to sorghum and sugarcane. Mitotic FISH of six different subclones of BAC 22B2 showed that pCEN38 produced the strongest specificity to the A(b) subgenome and signal occurred primarily near centromeres. Southern blots of pCEN38 to 21 crop plants revealed a narrow taxonomic distribution. Meiotic metaphase I FISH positioned pCEN38 sequences near active centromeres. Pachytene FISH revealed that the distributions are trimodal in several B(b) and possibly all sorghum chromosomes. DNA sequencing revealed that the pCEN38 fragment contains three tandemly repeated dimers (<280 bp) of the same sequence family found in sorghum clone pSau3A10, and that each dimer consists of two divergent monomers (<140 bp). Sequence comparisons revealed homology between the pCEN38 monomers and the SCEN 140 bp tandem repeat family of sugarcane. FISH of pCEN38 yielded signal in centromere regions of most but not all sugarcane chromosomes. Results suggest that sugarcane and sorghum share at least one ancestor harboring elements similar to pCEN38 and SCEN and that each species had an ancestor in which the repetitive element was weakly present or lacking.     

Localization of a new highly repeated DNA sequence of Lemur cafta (Lemuridae, Strepsirhini).

We have isolated and cloned an 800-bp highly repeated DNA (HRDNA) sequence from Lemur catta (LCA) and described its localization on LCA chromosomes. Lemur catta HRDNA sequences were localized by performing FISH experiments on standard and elongated metaphasic chromosomes using an LCA HRDNA probe (LCASAT). A complex hybridization pattern was detected. A strong pericentromeric hybridization signal was observed on most LCA chromosomes. Chromosomes 7 and 13 were lit in pericentromeric regions, as well as in the interspersed heterochromatin. Chromosomes 1, 3, 4, 17, 19, X, and microchromosomes (20, 25, 26, and 27) showed no signals in the pericentromeric region, but chromosomes 3 and 4 showed a positive hybridization in heterochromatic regions. The 800-bp L catta HRDNA was species specific. We performed FISH experiments with the LCASAT probe on Eulemur macaco macaco (EMA) and Eulemur fulvus fulvus (EFU) metaphases and no positive signal of hybridization was detected. These findings were also confirmed by Southern blot analysis and PCR.     

Molecular characterization and chromosomal localization of two families of satellite DNA in Prochilodus lineatus (Pisces,Prochilodontidae), a species with B chromosomes

Prochilodus lineatus, an abundant species in the Mogi-Guaçu river basin, represents a large part of the region's fishing potential. Karyotypic analyses based on classic cytogenetic techniques have revealed the presence of 54 meta-submetacentric type chromosomes, together with the occurrence of small supernumerary chromosomes with intra and interindividual variations. This paper describes the genomic organization of two families of satellite DNA in the P. lineatus genome. The chromosomal localization these two repetitive DNA families through fluorescence in situ hybridization (FISH) demonstrated that the SATH1 satellite DNA family, composed of approximately 900 bp, was located in the pericentromeric region of a group of chromosomes of the standard complement, as well as on all the B chromosomes. The SATH2 satellite family has a monomeric unit of 441 bp and was located in the pericentromeric regions of some chromosomes of the standard complement, but was absent in the B chromosomes. Double FISH analyses showed that these two families participate jointly in the pericentromeric organization of several chromosomes of this species. The data obtained in this study support the hypothesis that the B chromosomes derive from chromosomes of the standard complement, which are carriers of the SATH1 satellite DNA.     

Characterization and localization of repetitive DNA sequences in the ornamental Alstroemeria aurea Graham

 Three repetitive DNA sequences were isolated from a genomic DNA library of the ornamental Alstroemeria aurea Graham. Two repeats, A001-I and A001-II, were quite homologous and highly A. aurea-specific. A001-I was a 217-bp sequence with several telomeric TTTAGGG repeats at the 5′ end and a unique sequence of 98 bp at the other end. The third repeat, A001-IV, was a 840-bp sequence which contained two sub-sequences of 56 and 74 bp respectively, previously found in chloroplast (cp) DNA of tobacco and spinach and to a lesser extent in the cpDNA of maize and rice. Repeat A001-IV was not species-specific and its hybridization signal was weaker than the other repeats. Fluorescence in situ hybridization (FISH) revealed the A. aurea-specific repeats to be located in the heterochromatic regions of all A. aurea chromosomes. The differences in FISH pattern make them useful tools for karyotype analysis. The non-species-specific sequence A001-IV gave a dispersed signal over all the Alstroemeria chromosomes in an interspecific hybrid. The potential use of these repetitive DNA sequences for the study of phylogenetic relationships within the genus Alstroemeria is discussed. Received: 24 November 1996/Accepted: 20 December 1996     

A single, recent origin of the accessory B chromosome of the grasshopper Eyprepocnemis plorans

B chromosomes are dispensable chromosomes found in >2000 eukaryotic species, usually behaving as genomic parasites. Most B chromosomes seem to be made up of the same kind of DNA sequences present in the A chromosomes. This sequence similarity makes it difficult to obtain specific molecular probes that may permit B-presence diagnosis without cytogenetic analysis. We have developed a sequence-characterized amplified region (SCAR) marker for B chromosomes in the grasshopper Eyprepocnemis plorans, which specifically amplifies a 1510-bp DNA fragment exclusively in B-carrying individuals. Fluorescent in situ hybridization and fiber FISH analyses showed that this marker is a tandemly repeated DNA sequence closely intermingled with 45S rDNA. PCR reactions showed the presence of SCAR-like sequences in the A chromosomes, but in two separate fragments, supporting the intraspecific origin of B chromosomes in this species. SCAR marker DNA sequence showed to be identical in B chromosome variants from several localities from Spain and Morocco, and it was very similar to those found in B chromosome variants from Greece and Armenia. This strongly suggests that this sequence was already present in the ancestral B chromosome of this species. In addition, the scarce sequence variation observed among several B variants from very distant populations suggests either a functional constraint or, more likely, a recent and unique origin for B chromosomes in this species.